Huntington’s disease
• 1 out of the 14 documented trinucleotide repeat disorders; 1 of several polyglutamine diseases
• The trinucleotide repeat in Huntington’s disease is the CAG repeat. Patients with the CAG repeat in the Huntingtin gene who have Huntington’s disease have repeat lengths between 39-70 repeats. A normal chromosome only has 9 to 30 CAG's.
• This repetition produces an altered form of the Htt protein, mutant Huntingtin which results in neuronal cell death in select areas of the brain and is a terminal illness.
• Since the Huntingtin protein is found in most neurons throughout the brain, these neurons become infected and start a process of cell death. Once enough cells die within the brain, Huntington’s disease occurs.
• Symptoms: Loss of facial expression (called "masks in movement") or exaggerated facial gestures, ability to sit or stand stably, speech, chewing and swallowing. Eventually, it leads to an inability to walk, talk and eat. Death occurs generally 10- 30 years after the first sign of symptoms.
• Prevalence:1 in every 10,000 Americans have Huntington’s disease, with about 150,000 at risk of inheriting it from a parent.
• Currently, there is no cure, but symptoms are managed by using medications. Gene Technology
A case for basic science
The beginning of some of the most important science of this century started in the most unexpected place
Transitional generation
Bacteria and Bacteriophage
Bacteriophage are viruses that infect only bacteria
Hershey and Chase proved that DNA was the genetic material using bacteriophages (1940)
E. coli could "restrict" (prevent) infection from certain phage but not others---restriction (1960s)
Restriction Enzymes
An enzyme was isolated from E. coli that cut DNA in a specific pattern
The enzyme binds to a specific sequence of DNA (GAATTC) and cuts the DNA backbone on in a pattern that leaves a 4 base overhang or "sticky end"
Impact:
The ability to cut DNA at a known sequence opened the world of gene technology
Cloning
Plasmids--small circles of E. coli DNA
Restriction Endonucleases cut
Same sequence
Matching cuts
Allows DNA from different sources to be cut and then linked back together in new combinations
PLASMIDS ARE USED TO CLONE
DNA fingerprinting
Restriction Endonucleases cut DNA at the same sequence every time
This allows pieces of DNA cut with endonucleases to be separated on a gel
Same source of DNA--Same pattern of DNA pieces
A GENE is a segment of nucleic acid that contains the information necessary to produce a functional RNA product in a controlled manner.
An ALLELE (pronounced al-eel or al-e-ul) is any one of a number of viable DNA codings that occupies a given LOCUS (position) on a chromosome. Usually alleles are DNA (deoxyribonucleic acid) sequences that code for a gene, but sometimes the term is used to refer to a non-gene sequence.
in humans, most cells are DIPLOID (containing one set of chromosomes from each parent, two sets in all), but SEX CELLS (sperm and egg) are HAPLOID (containing one set of chromosomes)
Every gene in a diploid organism has two alleles at the gene's locus. These alleles are defined as DOMINANT or RECESSIVE, depending on the phenotype resulting from the two alleles. If a gene's two alleles are both dominant or both recessive, that specific gene is HOMOZYGOUS. If one allele is dominant and the other is recessive, the gene is HETEROZYGOUS.
A ZYGOTE is a cell that is the result of fertilization.
A GENOTYPE is the composition of part of an individual's genome which contributes to determining a specific trait
AA aa Ab
HOMOLOGOUS CHROMOSOMES are non-identical chromosomes that can pair (synapse) during meiosis, and are believed to share common ancestry.
PHENOTYPE-the look of something. Which trait shows up.
Genetic engineering/Gene Tech:
Tools and Techniques
1. Restriction Enzymes
2. Gel electrophoresis
3. Cloning
4. Polymerase chain reaction (PCR)
5. DNA sequencing
6. Pedigree Analysis
Applications:
1. Recombinant DNA
2. Transgenic organisms
3. Gene therapy
4. DNA fingerprinting
5. Human Genome project
6. Stem cell research
Gel Electrophoresis
Used in: forensics, gene determination
Separates DNA fragments by size creating a "fingerprint"
1. DNA fragments put in a gel
2. Electrical current applied
3. DNA (negative charge) migrates
4. Creates banding pattern
Cloning=genetically identical progeny
Therefore, cloning is any technique used to grow or produce genetically identical organisms like bacteria, stem cells, cell culture, and animals
Recombinant DNA
Combining the DNA from 2 different organisms with the aid of restriction enzymes
Cloning vector is often a bacterial plasmid
-Fast reproduction rate
-Make multiple copy of cloned gene
-To make large quantities of a particular substance coded for by inserted gene. (ex: Human insulin)
Polymerase Chain Reaction (PCR)
Exponential amplification
Amplify(copy) DNA sequences --> 1-100 billion in a few hours
DNA sample mixed w/ DNA polymerase and nucleotide primers
Mixture cycles through temperature changes
Changing temperature causes the mixture to alternate between DNA melting and DNA replication
DNA sequencing
Process used to determine exact order of bases in DNA
Use specially designed bases that 1. stop polymerization at random places 2. flourescent dyes specific for each base
Genetic relatedness
Phylogenic tree of grouse based on mitochondrial DNA
Applicant: Recombinant Insulin
1. Humulin
2. Example of a recombinant protein, used medically
3. Previous to 1982, most insulin was purified pig insulin
DNA fingerprinting
1. Alec Jefferies (England) 1984
2. Originally used similar technique to look for markers from certain genetic disorders
3. Principle-everyone has different amounts of non-coding DNA and therefore unique patterns of DNA bands
Use the restriction enzyme to cut DNA at specific sequences
Gel electrophoresis separates the pieces of DNA by size
Each source of DNA will have a characteristic pattern of DNA pieces
Samples can be taken from:
1. Solve crimes (murder, rape)
2. Paternity cases
3. Help endangered species
4. Evolutionary studies
Pedigree Analysis
Traces genetic traits through generations. Facilitates detection of inheritance patterns
When a disease/trait effects more than one member of a mily
Application:
1. Recombinant DNA
2. Human Genomre project
3. Transgenic organism
4. Gene therapy
5. Karyotyping and FISH
6. Microarrays
7. Stem cell research
6. Microarrays, 3 major points:
- Difference between genetics and genomics: Genetics studies one gene at a time, genomics studies many genes at a time.
- Measuring gene expression: In an organism, some genes are turned off and some are turned on. This is what we try to see in genetics and genomics
- Structure/Function: DNA microarrays are used in genomics, not genetics. Each chip has thousands of spots, each one representing a gene.
Stem Cells
1. Stem cells can become basically any type of cell in the body.
2. There are 3 main types of stem cells:
a. Umbilicord blood
b. Embryonic
-Embryonic stem cells are derived from blastocysts. Blastocysts consists of 70-100 cells. There are two layers in blastocysts: an inner layer and an outer layer. The inner layer forms forms the fetus and the outer layer goes on to form the placenta. Embryonic stem cells are taken from the inner layer of a blastocyst.
c. Adult Stem cells
3. Embryonic Stem cells are debated about because many conservatives do not believe in destroying embryoes in order to save already existant human life.
Karyotyping in Fluorescent in Situ Hybridization (FISH)
1.FISH is:
-a laboratory technique used to find the number of copies of a certain segment of DNA present in a cell
-can identify structurally-abnormal chromosomes
2. FISH is used for:
- finding specific features in DNA. These features help in genetic counseling, medicine, and species identification
3. Flouresence :
-a part of DNA is chemically enhanced and labeled to make it appear very brightly colored
- to help organize the pairs into a karyotype they are dyed with fluorescent dye to show up under a microscope.
4.Karyotyping:
-A karyotype is a man made arrangement of all the chromosomes in a cell
-Used to determine aspects of a persons genotype
The Steps of GENE SILENCING, a method of "silencing" or "blocking" certain genetic codes:
• Step 1: scientist incerts a double-stranded RNA into a cell, one of the strands of that RNA contains the identical sequence of bases to the gene that the scientist wants to silence (for now we will call it code X)
• Step 2: because cells should only have single-stranded RNA the cell identifies the double-stranded RNA as an intruder
• Step 3: it is the Dicer enzyme’s job to get rid of this intruder RNA strand and then goes after the double-stranded RNA, however, when it does this, because one of the stands in the double-stranded RNA codes for the gene that is desired to be silent (code X), and there is an identical code for that same gene in the cell’s original mRNA (also code X), the Dicer enzyme recognizes both code Xs as intruders and destroys the double-stranded RNA along with the code X portion of the cell’s original mRNA
• Step 4: because the Dice enzyme destroyed the code X in the cell’s mRNA code X will no longer be translated through protein synthesis and will not code to make the undesired protein, and therefore it will never be expressed
History
The national institute of health and the U.S department of energy collaborated and began to plan the human genome project
It was formally started in 1989
The two agencies formed common working groups on mapping information as well as working on social, ethical and legal implications of genome research.
The human genome organization was formed in 1988. It mediates international collaboration about genomics.
Goals of the HGP
Identify all the approximately 20,000-25,000 genes in human DNA
Generate genetic linkage and physical maps to cover all the human chromosomes
Determine the sequences of the 3 billion chemical base pairs that make up human DNA
Store this information in databases
Improve tools for data analysis
Transfer related technologies to the private sector
Address the ethical, legal and social issues that may arise from the project.
Objectives
1. Develop accurate maps of the human genome and genomes of several other well-known organisms
2. To determine the complete base sequences of genomes
“genome of an organism is its whole hereditary information and is encoded in the DNA”
Techniques
There are several techniques used to help scientists determine the exact location of a gene
Techniques may allow scientists to learn the precise location of genes or to determine base sequences
Whole Genome-shotgun
Breaks the genomes into smaller pieces; about 150,000 base pairs in length
The new smaller pieces are called bacterial artificial chromosomes
This is because they can be put into bacteria where they are copied by the bacterial DNA replication machinery
Then each of the pieces are individually sequences as a small “shotgun” project
All the separate chunks are then stitched back together to form a chromosome
Cytogenetics
This technique allows researches to examine individual chromosomes
Uses various staining methods to identify each individual chromosome
One method is called banding
Banding is done by breaking open nuclei from dividing cells and then putting the chromosomes on a slide
The chromosome is then stained and looked at through a microscope
A dark band appears in characteristic positions on different chromosomes and detect abnormalities
In situ hybridization
Just like with cytogenetics, researchers prepare cells so they can distinguish each chromosome from one another
They then use an isolated fragment of radioactively-tagged DNA from the gene under investigation as a probe
They probe sticks to the chromosome that has the complementary sequence
This reveals the site of the gene from which the probe came
Genetic linkage analysis
Allows scientists to track a gene and determine its exact location
It doesn’t require cloning of duplicating the gene
Since chromosomes are inherited, the genes on the chromosome are inherited as well. Genes can separate by recombination during meiosis but the closer the genes are to each other then the most likely they are linked and won’t separate
Southern Analysis
Allows researchers to identify a particular DNA fragment within a large collection of unrelated sequences
1. The DNA restriction enzyme cuts DNA
2. The different sized DNA fragments are separated by gel electrophoresis
The fragments are then transferred from the gel onto a nylon membrane
4. They expose the fragment on the membrane to a radioactive probe which hybridize to any complementary DNA sequence
Yeast artificial chromosomes
Allows scientists to borrow DNA duplication machinery of cells
Before YAC researchers had to use E. coli bacteria to make clones of pieces of DNA. The clone could only hold about 50,000 nucleotides
With the development of YACs, scientists can clone up to 1 million nucleotides
The developed of YACS has made it possible to put large pieces of DNA into yeast cells and reproduce them in very large quantitites
Benefits
Advances in medicine and biotechnology
Test predicting the genetic probability of breast cancer, disorders of homeostasis, cystic fibrosis, liver disease and may other. Also, etiologies fro cancer and Alzheimer's disease are likely to benefit
Narrowing research to a single gene helps scientists to easily find the effect of the gene
Analyzing DNA sequences of different organisms can help with the study of the theory of evolution.
THE END
Gene Therapy
Methods:
There are many different methods for inserting the gene into the cell
Differences between methods are usually in the vector
In all cases the vector takes the gene that has been cut using the restriction enzyme and places into the DNA of the cell
Examples-
Viral: Viruses are used as the vector. They infect the area that is affected by the defective gene and insert the “good” gene into the cells there
Direct: The DNA is directly inserted into the cells. Unfortunately this method can only be used with certain tissues. It also requires large amounts of DNA
Methods (cont.)
Lipid Sphere: DNA is inserted into an artificial lipid sphere. This sphere can pass through the membrane and into the cells where it inserts the gene.
Linking: The DNA is linked to a molecule that will bond to cell receptors. The DNA enters the cell once it has bound to the receptors and inserts the gene. This method is usually not as effective.
Background:
1980s: Scientists discovered how to clone and sequence DNA. With this knowledge, laboratory scientists could isolate and extract specific DNA segments in the human genome.
With this knowledge, laboratory scientists could isolate and extract specific DNA segments in the human genome.
-Researchers applied this knowledge to correct defective genes responsible for mutation development.
-Mutated segments of DNA were removed and replaced with normal fractions of DNA, extracted from the same region of another organism.
-Ultimately, this would ameliorate mutated genes and allow for a proper construction of proteins because the order of base pairs determines the construction of proteins.
First Procedure:
9/14/1990: Brandon Rogers performed the first, approved gene therapy procedure.
Patient: Ashanti DeSilva
-DeSilva was born with a rare genetic disease, known as Severe Combined Immunodeficiency (SCID.)
Most patients die before adulthood through an infection that the immune system cannot stop.
Procedure:
1. Doctor’s removed white blood cells from her body and let them grow in a lab.
2. The missing gene was inserted into the genetically modified cells.
3. Then they were transported back into the patients bloodstream.
Results of Ashanti’s Procedure
-The procedure was not a cure, since white blood cells only work for a few months.
-This process must be repeated once the white blood cells run out, in order for her to have a strong immune system.
Advances/ For the Future:
Recently Gene Therapy has been used to help to treat certain kinds of cancer, and as time goes on we could treat all kinds of cancer.
Like an organ, the new genes placed inside the patient, the body can reject it. Scientist found out that by putting microRNA on the gene, the body wouldn’t recognize the gene as an invade and it can survive.
In 2003, scientists used liposomes covered in polyethylene glycol to insert the genes into the brain, this was a big step in Gene Therapy.
Over the years, gene therapy has been used in myeliod cells (bone marrow), the brain, and soon many other parts of the body can successfully be treated with gene therapy.
Ethics:
Though gene therapy may be seen as the future to curing genetic disorders, there is a large faction morally opposed to the technique. A boy named Jesse Gelsinger was born June 18, 1981 suffering from ornthine transcarbamylase deficiency, a genetic condition where the liver cannot break down ammonia in cells, a byproduct from protein breakdown. Usually this disease is inherited and infants die soon after birth, however Jesse’s case was from a genetic mutation and some of his body cells worked normally. Jessie would have had to live on several medications and a resticted diet. On September 13, 1999 adenoviruses were injected into Jesse’s body with the corrected gene and scientists hoped that it would correct the defects; however, Jesse died September 17 from a massive immune response to the virus scientists had hoped would correct his disorder. Obviously gene therapy has its risks, and many believe human lives are not worth wasting. The Council for Responsible Genetics writes that the US government has publicized gene therapy too much without proving its successes. It also feels that too much experimentation is being directed towards curing and trying to prevent cancer. In fact, if the wrong gene is mutated as a result of gene therapy, cancerous cells can develop. Also, there are concerns that the virus used to implant the corrected gene can somehow regain its infection quality and infect the patient with the virus.
IMPORTANT VOCABULARY:
1. Bacteriophage- viruses that infect only bacteria 2.DIPLOID-containing one set of chromosomes from each parent, two sets in all 3.HAPLOID-containing one set of chromosomes 4.HOMOLOGOUS CHROMOSOMES -- non-identical chromosomes that can pair (synapse) during meiosis, and are believed to share common ancestry. 5. PHENOTYPE-the look of something. Which trait shows up. 6. Pedigree Analysis-Traces genetic traits through generations.
Transgenic Animals:
lDefinition: An animal which has had its genome altered to contain a gene from a foreign species, resulting in the animal incorporating the gene into its DNA and
Uses
lAgricultural
lBreeding: By isolating traits to specific genes, animals can be injected with more optimum genes, allowing farmers to have a more profitable trade
lQuality: Genes can be injected to reduce lactose or cholesterol in milk, produce more meaty pigs and cattle, make sheep grow more wool, etc.
lDisease Resistance: Some genes create a resistance to certain diseases, and at present scientists are working on influenza-resistant pigs
lMedical
lXenotransplantation: This process involves transplantation of one species’ organs into another. Animals such as pigs may be able to provide donor organs.
lPharmaceuticals: Insulin, growth horomnes, and blood anit-clotting factors have already been made and collected from the milk of cows, sheep, or goats.
lNutritional Supplements: Cows can and already have been made to produce human protein-enriched milk. This milk is much healthier, and could be given to the newborn, weak, or elderly.
lHuman Gene Therapy: By adding a normal copy of a gene to a person with a defective one, some of the mutations’ effects could be reversed.
lMedical (Cont.)
lDisease Testing: Using transgenic methods animals can be given human genes. This allows the animals to be infected by human diseases that they would otherwise be unaffected on. Scientists can then test treatments on the animals without having to use actual humans.
lKnockout Mice: Some mice can be produced to be homozygous for a nonfunctional gene, literally causing it to be “knocked out.” Mice aren’t affected by this, and the gene is often pleiotropic (affects many different areas in many different ways). Because of this these mice can be used to determine to uses of certain genes.
lIndustrial Applications
lCertain animal genes can be combined in order to create better materials.
lEx) Nexia Biotechnologies placed spider genes into the cells of a goat, causing the animal to manufacture milk with tiny silk strands in it. When extracted and woven together, the strands produce a light, tough, and flexible material tha can be used for things such as military uniforms and tennis racket strings.
lSome see it as messing around with the natural order of things
lModified organisms could cross bread with non modified organisms and cause future unforeseeable consequences
lPossible creation of new allergens or toxins - Some fruits carry antibiotic genes, and the consumption of these fruits could lead to the decreased effect of antibiotics
Negatives
lMonarch butterflies became stunted and died when they ate pollen from genetically modified corn
lTransgenic animals suffer more abnormalities than regular research animals
lMany animals receive effects such as reduced fertility
lThis new technology raises many ethical questions:
lShould overall guidelines be created for transgenesis?
lShould animal welfare be entirely ignored?
lShould in vitro processes be used instead of live animals?
lWill transgenesis have an effect on basic life processes such as evolution?
Huntington’s disease
• 1 out of the 14 documented trinucleotide repeat disorders; 1 of several polyglutamine diseases
• The trinucleotide repeat in Huntington’s disease is the CAG repeat. Patients with the CAG repeat in the Huntingtin gene who have Huntington’s disease have repeat lengths between 39-70 repeats. A normal chromosome only has 9 to 30 CAG's.
• This repetition produces an altered form of the Htt protein, mutant Huntingtin which results in neuronal cell death in select areas of the brain and is a terminal illness.
• Since the Huntingtin protein is found in most neurons throughout the brain, these neurons become infected and start a process of cell death. Once enough cells die within the brain, Huntington’s disease occurs.
• Symptoms: Loss of facial expression (called "masks in movement") or exaggerated facial gestures, ability to sit or stand stably, speech, chewing and swallowing. Eventually, it leads to an inability to walk, talk and eat. Death occurs generally 10- 30 years after the first sign of symptoms.
• Prevalence:1 in every 10,000 Americans have Huntington’s disease, with about 150,000 at risk of inheriting it from a parent.
• Currently, there is no cure, but symptoms are managed by using medications.
Gene Technology
A case for basic science
A GENE is a segment of nucleic acid that contains the information necessary to produce a functional RNA product in a controlled manner.
An ALLELE (pronounced al-eel or al-e-ul) is any one of a number of viable DNA codings that occupies a given LOCUS (position) on a chromosome. Usually alleles are DNA (deoxyribonucleic acid) sequences that code for a gene, but sometimes the term is used to refer to a non-gene sequence.
in humans, most cells are DIPLOID (containing one set of chromosomes from each parent, two sets in all), but SEX CELLS (sperm and egg) are HAPLOID (containing one set of chromosomes)
Every gene in a diploid organism has two alleles at the gene's locus. These alleles are defined as DOMINANT or RECESSIVE, depending on the phenotype resulting from the two alleles. If a gene's two alleles are both dominant or both recessive, that specific gene is HOMOZYGOUS. If one allele is dominant and the other is recessive, the gene is HETEROZYGOUS.
A ZYGOTE is a cell that is the result of fertilization.
A GENOTYPE is the composition of part of an individual's genome which contributes to determining a specific trait
AA aa Ab
HOMOLOGOUS CHROMOSOMES are non-identical chromosomes that can pair (synapse) during meiosis, and are believed to share common ancestry.
PHENOTYPE-the look of something. Which trait shows up.
Genetic engineering/Gene Tech:
Tools and Techniques
1. Restriction Enzymes
2. Gel electrophoresis
3. Cloning
4. Polymerase chain reaction (PCR)
5. DNA sequencing
6. Pedigree Analysis
Applications:
1. Recombinant DNA
2. Transgenic organisms
3. Gene therapy
4. DNA fingerprinting
5. Human Genome project
6. Stem cell research
Gel Electrophoresis
Used in: forensics, gene determination
Separates DNA fragments by size creating a "fingerprint"
1. DNA fragments put in a gel
2. Electrical current applied
3. DNA (negative charge) migrates
4. Creates banding pattern
Cloning=genetically identical progeny
Therefore, cloning is any technique used to grow or produce genetically identical organisms like bacteria, stem cells, cell culture, and animals
Recombinant DNA
Combining the DNA from 2 different organisms with the aid of restriction enzymes
Cloning vector is often a bacterial plasmid
-Fast reproduction rate
-Make multiple copy of cloned gene
-To make large quantities of a particular substance coded for by inserted gene. (ex: Human insulin)
Polymerase Chain Reaction (PCR)
Exponential amplification
Amplify(copy) DNA sequences --> 1-100 billion in a few hours
DNA sample mixed w/ DNA polymerase and nucleotide primers
Mixture cycles through temperature changes
Changing temperature causes the mixture to alternate between DNA melting and DNA replication
DNA sequencing
Process used to determine exact order of bases in DNA
Use specially designed bases that 1. stop polymerization at random places 2. flourescent dyes specific for each base
Genetic relatedness
Phylogenic tree of grouse based on mitochondrial DNA
Applicant: Recombinant Insulin
1. Humulin
2. Example of a recombinant protein, used medically
3. Previous to 1982, most insulin was purified pig insulin
DNA fingerprinting
1. Alec Jefferies (England) 1984
2. Originally used similar technique to look for markers from certain genetic disorders
3. Principle-everyone has different amounts of non-coding DNA and therefore unique patterns of DNA bands
Use the restriction enzyme to cut DNA at specific sequences
Gel electrophoresis separates the pieces of DNA by size
Each source of DNA will have a characteristic pattern of DNA pieces
Samples can be taken from:
1. Solve crimes (murder, rape)
2. Paternity cases
3. Help endangered species
4. Evolutionary studies
Pedigree Analysis
Traces genetic traits through generations. Facilitates detection of inheritance patterns
When a disease/trait effects more than one member of a mily
Application:
1. Recombinant DNA
2. Human Genomre project
3. Transgenic organism
4. Gene therapy
5. Karyotyping and FISH
6. Microarrays
7. Stem cell research
6. Microarrays, 3 major points:
- Difference between genetics and genomics: Genetics studies one gene at a time, genomics studies many genes at a time.
- Measuring gene expression: In an organism, some genes are turned off and some are turned on. This is what we try to see in genetics and genomics
- Structure/Function: DNA microarrays are used in genomics, not genetics. Each chip has thousands of spots, each one representing a gene.
Stem Cells
1. Stem cells can become basically any type of cell in the body.
2. There are 3 main types of stem cells:
a. Umbilicord blood
b. Embryonic
-Embryonic stem cells are derived from blastocysts. Blastocysts consists of 70-100 cells. There are two layers in blastocysts: an inner layer and an outer layer. The inner layer forms forms the fetus and the outer layer goes on to form the placenta. Embryonic stem cells are taken from the inner layer of a blastocyst.
c. Adult Stem cells
3. Embryonic Stem cells are debated about because many conservatives do not believe in destroying embryoes in order to save already existant human life.
Karyotyping in Fluorescent in Situ Hybridization (FISH)
1.FISH is:
-a laboratory technique used to find the number of copies of a certain segment of DNA present in a cell
-can identify structurally-abnormal chromosomes
2. FISH is used for:
- finding specific features in DNA. These features help in genetic counseling, medicine, and species identification
3. Flouresence :
-a part of DNA is chemically enhanced and labeled to make it appear very brightly colored
- to help organize the pairs into a karyotype they are dyed with fluorescent dye to show up under a microscope.
4.Karyotyping:
-A karyotype is a man made arrangement of all the chromosomes in a cell
-Used to determine aspects of a persons genotype
The Steps of GENE SILENCING, a method of "silencing" or "blocking" certain genetic codes:
• Step 1: scientist incerts a double-stranded RNA into a cell, one of the strands of that RNA contains the identical sequence of bases to the gene that the scientist wants to silence (for now we will call it code X)
• Step 2: because cells should only have single-stranded RNA the cell identifies the double-stranded RNA as an intruder
• Step 3: it is the Dicer enzyme’s job to get rid of this intruder RNA strand and then goes after the double-stranded RNA, however, when it does this, because one of the stands in the double-stranded RNA codes for the gene that is desired to be silent (code X), and there is an identical code for that same gene in the cell’s original mRNA (also code X), the Dicer enzyme recognizes both code Xs as intruders and destroys the double-stranded RNA along with the code X portion of the cell’s original mRNA
• Step 4: because the Dice enzyme destroyed the code X in the cell’s mRNA code X will no longer be translated through protein synthesis and will not code to make the undesired protein, and therefore it will never be expressed
HUMAN GENOME PROJECT:Human Genome Project
A good link with all this information is http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml
History
The national institute of health and the U.S department of energy collaborated and began to plan the human genome project
It was formally started in 1989
The two agencies formed common working groups on mapping information as well as working on social, ethical and legal implications of genome research.
The human genome organization was formed in 1988. It mediates international collaboration about genomics.
Goals of the HGP
Identify all the approximately 20,000-25,000 genes in human DNA
Generate genetic linkage and physical maps to cover all the human chromosomes
Determine the sequences of the 3 billion chemical base pairs that make up human DNA
Store this information in databases
Improve tools for data analysis
Transfer related technologies to the private sector
Address the ethical, legal and social issues that may arise from the project.
Objectives
1. Develop accurate maps of the human genome and genomes of several other well-known organisms
2. To determine the complete base sequences of genomes
“genome of an organism is its whole hereditary information and is encoded in the DNA”
Techniques
There are several techniques used to help scientists determine the exact location of a gene
Techniques may allow scientists to learn the precise location of genes or to determine base sequences
Whole Genome-shotgun
Breaks the genomes into smaller pieces; about 150,000 base pairs in length
The new smaller pieces are called bacterial artificial chromosomes
This is because they can be put into bacteria where they are copied by the bacterial DNA replication machinery
Then each of the pieces are individually sequences as a small “shotgun” project
All the separate chunks are then stitched back together to form a chromosome
Cytogenetics
This technique allows researches to examine individual chromosomes
Uses various staining methods to identify each individual chromosome
One method is called banding
Banding is done by breaking open nuclei from dividing cells and then putting the chromosomes on a slide
The chromosome is then stained and looked at through a microscope
A dark band appears in characteristic positions on different chromosomes and detect abnormalities
In situ hybridization
Just like with cytogenetics, researchers prepare cells so they can distinguish each chromosome from one another
They then use an isolated fragment of radioactively-tagged DNA from the gene under investigation as a probe
They probe sticks to the chromosome that has the complementary sequence
This reveals the site of the gene from which the probe came
Genetic linkage analysis
Allows scientists to track a gene and determine its exact location
It doesn’t require cloning of duplicating the gene
Since chromosomes are inherited, the genes on the chromosome are inherited as well. Genes can separate by recombination during meiosis but the closer the genes are to each other then the most likely they are linked and won’t separate
Southern Analysis
Allows researchers to identify a particular DNA fragment within a large collection of unrelated sequences
1. The DNA restriction enzyme cuts DNA
2. The different sized DNA fragments are separated by gel electrophoresis
The fragments are then transferred from the gel onto a nylon membrane
4. They expose the fragment on the membrane to a radioactive probe which hybridize to any complementary DNA sequence
Yeast artificial chromosomes
Allows scientists to borrow DNA duplication machinery of cells
Before YAC researchers had to use E. coli bacteria to make clones of pieces of DNA. The clone could only hold about 50,000 nucleotides
With the development of YACs, scientists can clone up to 1 million nucleotides
The developed of YACS has made it possible to put large pieces of DNA into yeast cells and reproduce them in very large quantitites
Benefits
Advances in medicine and biotechnology
Test predicting the genetic probability of breast cancer, disorders of homeostasis, cystic fibrosis, liver disease and may other. Also, etiologies fro cancer and Alzheimer's disease are likely to benefit
Narrowing research to a single gene helps scientists to easily find the effect of the gene
Analyzing DNA sequences of different organisms can help with the study of the theory of evolution.
THE END
Gene Therapy
Methods:
There are many different methods for inserting the gene into the cell
Differences between methods are usually in the vector
In all cases the vector takes the gene that has been cut using the restriction enzyme and places into the DNA of the cell
Examples-
Viral: Viruses are used as the vector. They infect the area that is affected by the defective gene and insert the “good” gene into the cells there
Direct: The DNA is directly inserted into the cells. Unfortunately this method can only be used with certain tissues. It also requires large amounts of DNA
Methods (cont.)
Lipid Sphere: DNA is inserted into an artificial lipid sphere. This sphere can pass through the membrane and into the cells where it inserts the gene.
Linking: The DNA is linked to a molecule that will bond to cell receptors. The DNA enters the cell once it has bound to the receptors and inserts the gene. This method is usually not as effective.
Background:
1980s: Scientists discovered how to clone and sequence DNA. With this knowledge, laboratory scientists could isolate and extract specific DNA segments in the human genome.
With this knowledge, laboratory scientists could isolate and extract specific DNA segments in the human genome.
-Researchers applied this knowledge to correct defective genes responsible for mutation development.
-Mutated segments of DNA were removed and replaced with normal fractions of DNA, extracted from the same region of another organism.
-Ultimately, this would ameliorate mutated genes and allow for a proper construction of proteins because the order of base pairs determines the construction of proteins.
First Procedure:
9/14/1990: Brandon Rogers performed the first, approved gene therapy procedure.
Patient: Ashanti DeSilva
-DeSilva was born with a rare genetic disease, known as Severe Combined Immunodeficiency (SCID.)
Most patients die before adulthood through an infection that the immune system cannot stop.
Procedure:
1. Doctor’s removed white blood cells from her body and let them grow in a lab.
2. The missing gene was inserted into the genetically modified cells.
3. Then they were transported back into the patients bloodstream.
Results of Ashanti’s Procedure
-The procedure was not a cure, since white blood cells only work for a few months.
-This process must be repeated once the white blood cells run out, in order for her to have a strong immune system.
Advances/ For the Future:
Recently Gene Therapy has been used to help to treat certain kinds of cancer, and as time goes on we could treat all kinds of cancer.
Like an organ, the new genes placed inside the patient, the body can reject it. Scientist found out that by putting microRNA on the gene, the body wouldn’t recognize the gene as an invade and it can survive.
In 2003, scientists used liposomes covered in polyethylene glycol to insert the genes into the brain, this was a big step in Gene Therapy.
Over the years, gene therapy has been used in myeliod cells (bone marrow), the brain, and soon many other parts of the body can successfully be treated with gene therapy.
Ethics:
Though gene therapy may be seen as the future to curing genetic disorders, there is a large faction morally opposed to the technique. A boy named Jesse Gelsinger was born June 18, 1981 suffering from ornthine transcarbamylase deficiency, a genetic condition where the liver cannot break down ammonia in cells, a byproduct from protein breakdown. Usually this disease is inherited and infants die soon after birth, however Jesse’s case was from a genetic mutation and some of his body cells worked normally. Jessie would have had to live on several medications and a resticted diet. On September 13, 1999 adenoviruses were injected into Jesse’s body with the corrected gene and scientists hoped that it would correct the defects; however, Jesse died September 17 from a massive immune response to the virus scientists had hoped would correct his disorder. Obviously gene therapy has its risks, and many believe human lives are not worth wasting. The Council for Responsible Genetics writes that the US government has publicized gene therapy too much without proving its successes. It also feels that too much experimentation is being directed towards curing and trying to prevent cancer. In fact, if the wrong gene is mutated as a result of gene therapy, cancerous cells can develop. Also, there are concerns that the virus used to implant the corrected gene can somehow regain its infection quality and infect the patient with the virus.
IMPORTANT VOCABULARY:
1. Bacteriophage- viruses that infect only bacteria
2.DIPLOID-containing one set of chromosomes from each parent, two sets in all
3.HAPLOID-containing one set of chromosomes
4.HOMOLOGOUS CHROMOSOMES -- non-identical chromosomes that can pair (synapse) during meiosis, and are believed to share common ancestry.
5. PHENOTYPE-the look of something. Which trait shows up.
6. Pedigree Analysis-Traces genetic traits through generations.
Transgenic Animals:
lDefinition: An animal which has had its genome altered to contain a gene from a foreign species, resulting in the animal incorporating the gene into its DNA and
Uses
lAgricultural
lBreeding: By isolating traits to specific genes, animals can be injected with more optimum genes, allowing farmers to have a more profitable trade
lQuality: Genes can be injected to reduce lactose or cholesterol in milk, produce more meaty pigs and cattle, make sheep grow more wool, etc.
lDisease Resistance: Some genes create a resistance to certain diseases, and at present scientists are working on influenza-resistant pigs
lMedical
lXenotransplantation: This process involves transplantation of one species’ organs into another. Animals such as pigs may be able to provide donor organs.
lPharmaceuticals: Insulin, growth horomnes, and blood anit-clotting factors have already been made and collected from the milk of cows, sheep, or goats.
lNutritional Supplements: Cows can and already have been made to produce human protein-enriched milk. This milk is much healthier, and could be given to the newborn, weak, or elderly.
lHuman Gene Therapy: By adding a normal copy of a gene to a person with a defective one, some of the mutations’ effects could be reversed.
lMedical (Cont.)
lDisease Testing: Using transgenic methods animals can be given human genes. This allows the animals to be infected by human diseases that they would otherwise be unaffected on. Scientists can then test treatments on the animals without having to use actual humans.
lKnockout Mice: Some mice can be produced to be homozygous for a nonfunctional gene, literally causing it to be “knocked out.” Mice aren’t affected by this, and the gene is often pleiotropic (affects many different areas in many different ways). Because of this these mice can be used to determine to uses of certain genes.
lIndustrial Applications
lCertain animal genes can be combined in order to create better materials.
lEx) Nexia Biotechnologies placed spider genes into the cells of a goat, causing the animal to manufacture milk with tiny silk strands in it. When extracted and woven together, the strands produce a light, tough, and flexible material tha can be used for things such as military uniforms and tennis racket strings.
lSome see it as messing around with the natural order of things
lModified organisms could cross bread with non modified organisms and cause future unforeseeable consequences
lPossible creation of new allergens or toxins - Some fruits carry antibiotic genes, and the consumption of these fruits could lead to the decreased effect of antibiotics
Negatives
lMonarch butterflies became stunted and died when they ate pollen from genetically modified corn
lTransgenic animals suffer more abnormalities than regular research animals
lMany animals receive effects such as reduced fertility
lThis new technology raises many ethical questions:
lShould overall guidelines be created for transgenesis?
lShould animal welfare be entirely ignored?
lShould in vitro processes be used instead of live animals?
lWill transgenesis have an effect on basic life processes such as evolution?